Linking the genotype with the phenotype

Chief Investigators: Bill Ballard
Mitochondria are the Dr Jekyll and Mr Hyde of life but the nature of their relationship with an organism’s phenotype remains to be determined

As Dr Jekyll, they are the “powerhouse of the cell” and produce 80% of our cell’s energy in the form of ATP, which is essential for life.

But, as the misanthropic Mr. Hyde, mitochondria produce reactive oxygen species (also known as free radicals) as a by-product of normal metabolism. Reactive oxygen species damage DNA, cell membranes and lipids and are proposed to be a major contributor to cell and organismal death.

Thus, a common feature across all animals, including humans, is that mitochondrial bioenergetics is linked to oxidative stress, but the nature of these relationships with the phenotype of the organism has yet to be properly defined.

Our studies will solve this important problem for Drosophila (Fig. 1), one of the major model systems in biology using the bioenergetic framework (Fig. 2).

 

 

Fig. 1 Drosophila simulans harbours three distinct mtDNA haplogroups that are non-randomly distributed around the globe. This non-random distribution implies that the mtDNA genome is under strong selection and or is locally adapted.

Fig. 2 Bioenergetic framework. Under state III phosphorylating conditions, the system is defined as a tripartite network. Under state IV conditions, it becomes a two-branch system of substrate oxidation and uncoupling because the phosphorylation branch is not active. ROS = Reactive Oxygen Species.

 

Mitochondrial DNA variation is associated with measurable differences in life history traits and mitochondrial metabolism in Drosophila


J. William O. Ballard, Richard G. Melvin, Subhash D. Katewa, & Koen Maas
Evolution (in press)


Recent studies have used a variety of theoretical arguments to show that mtDNA is rarely evolving as a strictly neutral marker and that selection is operating on the mtDNA of many species. However, the vast majority of researchers are not convinced by these arguments because data linking mtDNA variation with phenotypic differences are limited. We investigated sequence variation in the three mtDNA and nine nuclear genes (including all isoforms) that encode the 12 subunits of cytochrome c oxidase of the electron transport chain in Drosophila. We then studied cytochrome c oxidase activity as a key aspect of mitochondrial bioenergetics and four life history traits. Flies with siIII mtDNA had higher cytochrome c oxidase activity (Fig. 3) and were more starvation resistant. Flies harboring siII mtDNA recovered faster from cold coma (Fig. 4), had greater egg size, and fecundity. The data show that evolutionary shifts can involve changes in mtDNA despite the small number of genes encoded in the organelle genome.

 

Fig. 3 Mean cytochrome c oxidase activity (±SE) of each fly line. Flies harboring siII mtDNA have lower cytochrome c oxidase activity than those with siIII mtDNA. Males have lower cytochrome c oxidase activity than females. The horizontal bar denotes the group mean. Lines: à 2KY15, □ 2KY17, ○ 2KY18, ∆ 2KY21, ♦ 3KY10, ■ 3KY12, · 3KY14, ♦ 3KY20.

Fig. 4 Mean coma recovery time (±SE) of males (M) females (F) of each fly line. Flies harboring siII mtDNA recovered faster from a 16H cold shock at 0°C. Significantly, flies with this mtDNA type also had greater survival following cold shock. These data suggest that siII harboring flies are preadapted for colder climates. The horizontal bar denotes the group mean.
 

A candidate complex approach to study functional mtDNA changes: Sequence variation and quaternary structure modeling of Drosophila COX

Richard G. Melvin, Subhash D. Katewa, & J. William O. Ballard
Journal of Molecular Evolution (submitted)

A problem with studying evolutionary dynamics of mtDNA is that classical population genetic techniques cannot identify selected substitutions because of genetic hitchhiking. We circumvent this problem by employing a candidate complex approach to study sequence variation in cytochrome c oxidase (COX) genes within and among three distinct Drosophila simulans mtDNA haplogroups. Firstly, we determined sequence variation in complete coding regions for all COX mtDNA and nuclear loci and their isoforms. Second, we constructed a quaternary structure model of D. simulans COX (Fig. 5). Third, we predict that seven of nine amino acid changes in D. simulans mtDNA are likely to be functionally important. Of these seven, genetic crosses can experimentally determine the functional significance of three (Fig. 6). Further, we predict that two amino acid changes and a two amino deletion in nuclear encoded COX loci are likely to influence cytochrome c oxidase activity. The utility of the approach was confirmed by modeling the levy mutation in D. melanogaster.

 

Fig. 5 Quaternary structure model of cytochrome c oxidase from Drosophila simulans. Blue denotes fixed changes in siI, green fixed changes in siII and red denotes polymorphisms.

Fig. 6 Catalytic core of cytochrome c oxidase showing the three mtDNA encoded subunits COI, II, and III. COI is colored pink, COII is yellow, and COIII is brown. The siI mtDNA lineage has two fixed (blue) amino acid replacement polymorphisms in COI and one in COII. The siII subunits have two fixed amino acid replacements (green) in COII.